The present invention relates to DNA containing the prolipoprotein (PLP) signal peptidase gene, to isolation of such DNA from appropriate strains of Escherichia coli (hereinafter "E. coli "). to plasmids incorporating such DNA and to microorganisms transformed with said plasmids.
It is generally considered that many microorganisms. including E. coli, produce at least some of their proteins initially in the form of a precursor. This applies particularly to outer cell wall and periplasmic proteins. Such a precursor can comprise the desired protein attached, for example at its amino-terminus, to a signal peptide, signal peptidases, which cleave off such a signal peptide, are considered to play a key role in the process of protein secretion across the cytoplasmic membrane. Among the E. coli proteins, the major outer membrane lipoprotein (LP) is the most abundant in terms of the number of molecules and has many unique features. It is believed that the structural gene for lipoprotein in E. coli can be transcribed highly efficiently and have been made to use lipoprotein as an expressionn vector.
However, to date, there are relatively few cases where such an expression vector has been successfully used to produce large quantities of proteins. This is probably because these proteins are toxic to E. coli and either kill the E. coli cells or are degraded when the concentration exceeds a certain (low) level. The LP gene is structural and it might be thought that increasing the number of such genes in the microbial cell would lead to an increase in the protein product. However, even when cloned into a multi-copy plasmid, such as PBR322, successful expression of large quantities to the desired protein does not seem to be achieved. It is probable that this is because the prolipoprotein produced by expression of the Lp genes in large quantities kills the cells.
This problem could be overcome by causing the gene products to be secreted outside of the cytoplasm, namely in the periplasm or in a medium. If such products can be secreted outside the cytoplasm away from the metabolic system of the cell, even a protein harmful to the metabolism can be produced and decomposition of the desired proteins in the cell will not occur. An important element in such secretion is the PLP signal peptidase. If the quantity of PLP signal peptidase produced is not commensurate with the quantity of PLP or of PLP linked to another desired prorein, then the quantity of PLP in the cell will increase, thus leading to poisoning of the cell.
A means of increasing production of PLP signal peptidase is thus urgently needed in order to enable genetically engineered cells, particularly E. coli cells, to produce large quantities of other desirable proteins. Moreover, excretion of desired proteins outside the cytoplasm has another advantage. When producing proteins by fermentation, isolation and purification of the protein product is far easier and more efficient if the product is outside the cell, particularly in the culture medium, than it is if the protein product remains within the cell.
We have now successfully isolated a DNA segment coding for PLP signal peptidase from the chromasomal material of E. coli and have incorporated the segment into a plasmid and demonstrated successful expression of the desired signal peptidase when the resulting plasmid is introduced into a microorganism.